观察采用液氮保存GGTA1/β4GalNT2双基因敲除近交系五指山小型猪猪皮异种移植的效果。

采用冻存双基因敲除猪断层皮片及市售基因转染猪皮进行异种移植。切取双基因敲除猪皮后采用玻璃化方法储皮，使用时复温。受体为恒河猴，分为实验组和对照组，每组4只。于每只猴背部切除4块4 cm×4 cm的皮肤，形成全层皮肤切除模型，分别移植冻存双基因敲除猪皮和基因转染猪皮。术后不同时间点观察皮片成活情况，通过颜色、质地等评估皮片活力，留取标本进行病理检查；于术前及术后4、7、10、14、21 d取血清，检测IL-2、IL-4、IFN-γ、IL-12含量。

实验组猪皮存留于创面时间[10～21(14.5±3.4)d]长于对照组[7～14(10.6±2.7)d]，差异有统计学意义(t＝3.272，P＝0.004)。术后第4天两组皮片均活力良好；术后第7天，实验组2个皮片局部有小水疱，对照组6个皮片皮下有大水疱；术后第10天，实验组5个皮片被完全撕脱，6个皮片整体仍温暖、柔软，对照组4个皮片整体仍温暖、柔软；术后第12天，实验组和对照组分别有3个、1个皮片整体仍温暖、柔软；术后第14天，实验组1个皮片仍保持温暖、柔软，对照组皮片则全部失活。病理观察显示，两组术后第4天皮片组织形态均完整；术后第7天，实验组皮片表皮细胞结构完整，对照组皮片表皮层细胞数量减少，两组均可见部分皮片与基底床形成嫁接；术后第10天，实验组表皮层细胞数量减少，表皮与真皮交界面局部分离，对照组表皮层细胞数量较前进一步减少，仍可见部分皮片与基底床嫁接现象；术后第14天，实验组表皮层细胞数量明显减少，仍可见嫁接现象，对照组可见基底床炎细胞浸润。实验组术后第10天血清IL-2和IL-4水平、术前与术后第4天血清IFN-γ水平、术后第14天血清IFN-γ/IL-4高于对照组。

采用冻存GGTA1/β4GalNT2双基因敲除近交系五指山小型猪猪皮移植后皮片存留于创面情况良好，可获得较长的创面存留时间。

To observe the effect of xenotransplantation of GGTA1/β4GalNT2 double-gene knockout inbred line Wuzhishan miniature pig skin preserved in liquid nitrogen.

Cryopreserved double-gene knockout pig split skin and commercially gene transfected pig skin were used. The double-gene knockout pig skin was preserved by vitrification method, and the skin was thawed when used for xenotransplantation. The receptor were rhesus monkey, which were divided into experimental group and control group, with 4 animals in each group, and four pieces of 4 cm×4 cm skin were removed on each monkey′s back, forming a full-thickness skin excision model. Cryopreserved double-gene knockout pig skin and gene transfected pigs kin were transplanted respectively. The survival of the skin was observed at different time points after the operation, and the vitality of the skin was evaluated by color, texture, etc. And samples were taken for pathological examination. Serum was taken before operation and on the 4th, 7th, 10th, 14th and 21st days after operation to detect IL-2, IL-4, IFN-γ, and IL-12 content.

The survival time of pig skin in the experimental group was [10-21(14.5±3.4)d], significantly longer than [7-14(10.6±2.7)d] in the control group (t=3.272, P=0.004). The skin grafts of both groups survived well on the 4th day after operation. On the 7th day after operation, there were small blisters on 2 skin grafts in the experimental group, and big blisters on 6 skin grafts in the control group. On the 10th day after operation, 5 skin grafts in the experimental group were completely torn off, 6 and 4 skin grafts were still warm and soft in the experimental and control group respectively. On the 12th day after operation, 3 and 1 skin grafts in the experimental group and the control group were still warm and soft. On the 14th day after operation, one skin graft in the experimental group remained warm and soft, while all skin grafts lost viablity in the control group. Pathological observation showed that the histological morphology of the skin grafts in both groups were normal on the 4th day after operation. On the 7th day after operation, the structure of skin epidermal cells in the experimental group was complete, while the number of epidermal cells in the control group was reduced. In a part of the skin grafts in both groups, engraft could be seen between the skin and wound bed. On the 10th day after operation, the number of epidermal cells in the experimental group decreased, and the interface between epidermis and dermis was partially separated, while the number of epidermal cells in the control group was further reduced compared with before, and the phenomenon of engraft still existed. On the 14th day after operation, the number of epidermal cells was significantly reduced and the phenomenon of engraft still existed in the experimental group. While infiltration of inflammatory cells in the wound bed could be seen in the control group. Serum IL-2 and IL-4 levels on the 10th day after operation, serum IFN-γ before operation and on the 4th day after operation, and the IFN-γ/IL-4 on the 14th day after operation in the experimental group were higher than those in the control group.

Cryopreserved GGTA1/β4GalNT2 double-gene knockout inbred line Wuzhishan miniature pig skin survives well after transplantation, and can obtain a relatively long survival time.